The overarching goal of this project is to understand the mechanisms by which transepidermal inoculation of human skin with vaccinia (scarification) leads to a protective immune response to smallpox, and to use this knowledge to help develop vaccination strategies that are both safe and effective, particularly for patients who currently are not vaccination candidates (e.g., patients with atopic dermatitis). Studies will be performed using both human and murine model systems. The potential for vaccinia to productively infect both normal and atopic skin tissue, cells, and artificial skin constructs, will be assessed by vanous techniques. In collaboration with investigators from Project 1, both skin and blood will be sampled at various time points from vaccinated normal volunteers. We will test the validity of our paradigm of cutaneous immune response, wherein Langerhans cells containing virus fragments from infected epidermis migrate to draining lymph nodes and differentiate into potent mature dendritic cells and activate naive T cells. These T cells expand clonally and differentiate into central memory and skin-homing effector memory T cells. Effector memory T cells extravasate from dermal vessels at the vaccine site and enter the papillary dermis and epidermis. Central memory cells traffic into secondary lymphoid tissues and provide long-term immunologic memory. We will characterize key cellular and humoral elements of the protective immune response to variola generated as a result of these events. We will test the extent to which atopic dermatitis patients and normal volunteers vaccinated with MVA develop similar key elements of this protective response. In murine models, we will manipulate the cutaneous microenvironment with biological response modifiers and determine whether these maneuvers improve the immune response to vaccinia. Transgenic mice with targeted epidermal expression of cytokines and chemokines that influence dendritic cell migration and function will also be studied. The efficacy of vaccination will be assessed by testing resistance to vaccinia challenge in these mice after prior vaccination with MVA or vaccinia, with a major goal being to enhance vaccination efficiency. The Harvard Skin Disease Research Center has studied both innate and acquired immune response mechanisms in skin for more than 15 years. The resources of the HSDRC will provide a rich environment for these studies and will enhance collaborative interactions with investigators leading projects 1, 2, and 4.